Enhancing antitumor efficacy of CLDN18.2-directed antibody-drug conjugates through autophagy inhibition in gastric cancer

Claudin18.2 (CLDN18.2) is overexpressed in cancers of the digestive system, rendering it an ideal drug target for antibody-drug conjugates (ADCs). Despite many CLDN18.2-directed ADCs undergoing clinical trials, the inconclusive underlying mechanisms pose a hurdle to extending the utility of these agents. In our study, αCLDN18.2-MMAE, an ADC composed of an anti-CLDN18.2 monoclonal antibody and the tubulin inhibitor MMAE, induced a dose-dependent apoptosis via the cleavage of caspase-9/PARP proteins in CLDN18.2-positive gastric cancer cells. It was worth noting that autophagy was remarkably activated during the αCLDN18.2-MMAE treatment, which was characterized by the accumulation of autophagosomes, the conversion of autophagy marker LC3 from its form I to II, and the complete autophagic flux. Inhibiting autophagy by autophagy inhibitor LY294002 remarkably enhanced αCLDN18.2-MMAE-induced cytotoxicity and caspase-mediated apoptosis, indicating the cytoprotective role of autophagy in CLDN18.2-directed ADC-treated gastric cancer cells. Combination with an autophagy inhibitor significantly potentiated the in vivo antitumoral efficacy of αCLDN18.2-MMAE. Besides, the Akt/mTOR pathway inactivation was demonstrated to be implicated in the autophagy initiation in αCLDN18.2-MMAE-treated gastric cancer cells. In conclusion, our study highlighted a groundbreaking investigation into the mechanism of the CLDN18.2-directed ADC, focusing on the crucial role of autophagy, providing a novel insight to treat gastric cancer by the combination of CLDN18.2-directed ADC and autophagy inhibitor.


INTRODUCTION
Gastric cancer is one of the most common cancers of the digestive system [1].Due to insidious symptoms and inadequate screening systems, patients are often diagnosed in advanced stages when surgical resection becomes unfeasible.Chemotherapy, primarily based on platinum and fluoropyrimidine, stands as the mainstay treatment for advanced and metastatic gastric cancer [2,3].However, it encounters challenges such as drug resistance and adverse effects, leading to a median overall survival of about 12 months [4].Hence, there is an urgent imperative to explore novel therapeutics.
Antibody-drug conjugates (ADCs) involve the conjugation of an antibody and cytotoxic agents through cleavable linkers, proving to be a promising targeted therapy [5].The promising targets of ADCs to treat gastric cancer mainly include HER2, Claudin18.2(CLDN18.2), and EGFR [6][7][8][9].Among them, CLDN18.2 has garnered much attention due to its specific and high expression in gastric cancer.CLDN18.2, a member of the Claudin protein family, typically features four transmembrane domains and two extracellular loops [10,11].In normal tissues, CLDN18.2epitopes, though present, are typically concealed by tight junctions, impeding their exposure.However, the disruption of tight junctions by malignant tumors facilitates the binding of antibody drugs [12].Zolbetuximab, an IgG1-type antibody targeting CLDN18.2, has demonstrated efficacy and safety in an international phase III clinical trial for CLDN18.2-positive,HER2-negative, unresectable or metastatic gastric cancer [13].In this trial, the combined use of zolbetuximab and chemotherapy significantly reduced the risk of disease progression and mortality.The median progression-free survival for the zolbetuximab group was 10.61 months, compared to 8.67 months for the placebo group.The efficacy and safety of zolbetuximab underscored the potential of CLDN18.2 as an encouraging therapeutic target for gastric cancer.According to information from both clinicaltrials.gov,and the drug clinical trial registration and information publicity platform, a dozen ADCs targeting CLDN18.2 are currently undergoing clinical trials, including SKB315, RC118, ATG022, LM302, SYSA1801, IBI343, AZD0901, TQB2103, EO-3021, and JS107.Despite the progress in clinical trials, the underlying mechanisms of these agents are not yet fully understood, posing a significant challenge to expanding the scope of their application.
Autophagy is a critical intracellular degradation process in eukaryotic cells, responsible for the clearance of senescent proteins and malfunctioning organelles, thus maintaining cellular homeostasis [14,15].In cancer treatment, the role of autophagy is multifaceted and contingent upon tumor types and stages of tumorigenesis [16,17].On one hand, autophagy serves as a tumor-suppressive mechanism by eliminating integrant cellular components.On the other hand, it exhibits a cytoprotective role, facilitating cancer cell survival and adaptation to harsh microenvironments characterized by hypoxia and nutrient scarcity.Several studies have shown that certain drugs such as asparaginase, signal regulatory protein α-Fc and cisplatin used in cancer treatment can induce autophagy [18][19][20].The combination therapy of autophagy inhibitors with certain antitumor drugs can significantly enhance their efficacy [21][22][23].In non-small cell lung cancer models, autophagy inhibitor chloroquine significantly enhanced the effectiveness of erlotinib in inhibiting tumor growth and suppressing the JAK2/STAT3/VEGFA pathway [24].However, it remains unclear whether and how autophagy influences CLDN18.2-targetedADC therapy in gastric cancer, highlighting the need for further investigation.
In this study, we delved into the potential impacts of αCLDN18.2-MMAE on CLDN18.2-positivegastric cancer cells, and it was the first report that we found autophagy was triggered during αCLDN18.2-MMAE-inducedcaspase-dependent apoptosis in gastric cancer cells.We further elucidated the cytoprotective role and underlying mechanism of autophagy induced by αCLDN18.2-directedADCs.In addition, inhibiting autophagy by LY294002 significantly potentiates the in vivo antitumor effects of αCLDN18.2-MMAE.Our findings underscored the potential of combining CLDN18.2-directedADCs with autophagy inhibitors as a novel and encouraging therapeutic approach to treat gastric cancer.
Apoptosis was activated by αCLDN18.2-MMAE in CLDN18.2positivegastric cancer cells Considering apoptosis as a pivotal form of cell death, we extended our investigation into the ability of αCLDN18.2-MMAE to induce apoptosis specifically in CLDN18.2-positivegastric cancer cells.After co-incubating MKN45-CLDN18.2and SNU-601 cells with varying concentrations of αCLDN18.2-MMAE for 48 h, apoptosis was assessed.As depicted in Fig. 2A and B, a dose-dependent increase in Annexin V-positive tumor cells was observed, including both early apoptotic (Annexin V + /PI -) and late apoptotic (Annexin V + /PI + ) cell populations.Additionally, Western Blot was conducted to examine alterations of apoptosis-related protein within MKN45-CLDN18.2 and SNU-601 cells after 48 h of treatment.As illustrated in Fig. 2C and D, elevated expressions of cleaved caspase-9 and cleaved PARP were observed, indicative of the activation of apoptosis-related pathways.These findings collectively confirmed the capacity of αCLDN18.2-MMAEinducing apoptosis in CLDN18.2-positivegastric cancer cells.

αCLDN18.2-MMAE induced autophagy in CLDN18.2-positive gastric cancer cells
During the observation of cell morphology through electron microscopy, a significant accumulation of autophagic vesicles was unexpectedly discovered in CLDN18.2-positivegastric cancer cells after αCLDN18.2-MMAEtreatment (Fig. 3A).To further validate the induction of autophagy, Cyto-ID, a fluorescent dye specifically labeling autophagosomes, was employed.Similarly, confocal microscopy results showed enhanced green fluorescence within the αCLDN18.2-MMAEtreatedcells, akin to the positive control group treated with rapamycin (Figs.3B and S2).Additionally, Western Blot results indicated an increase in LC3-II expression levels and a decrease in SQSTM1 expression levels (Fig. 3C, D).In autophagy, LC3-I is modified by a ubiquitin-like system and subsequently coupled with phosphatidylethanolamine to form LC3-II. SQSTM1 acts as a bridge between the ubiquitinated proteins and LC3 proteins, ultimately targeted for degradation by autophagy.Therefore, an increase in the ratio of LC3-II to LC3-I expression and a decrease in SQSTM1 expression can indicate the occurrence of autophagy.Furthermore, to observe autophagic flux, Cyto-ID, Lyso-Tracker, and Hoechst 33342 were utilized to stain autophagosomes (green), lysosomes (red), and nuclei (blue), respectively.After a 12-hour treatment with αCLDN18.2-MMAE,notable autophagosomes appeared in both MKN45-CLDN18.2and SNU-601 cells.Additionally, red fluorescence was also present in MKN45-CLDN18.2cells, indicative of lysosomal activation.At 24 h, autophagosomes and lysosomes showed bright fluorescence and co-localization, suggesting that autophagosomes fused with lysosomes to form autolysosomes.At 48 h, the green fluorescence was visibly weakened while the red fluorescence remained strong, indicating autophagosome degradation (Fig. 3E, F).In conclusion, αCLDN18.2-MMAEinitiated both autophagosome formation and autophagic flux in CLDN18.2-positivegastric cancer cells.

Inhibiting autophagy reinforced cytotoxicity and apoptosis triggered by αCLDN18.2-MMAE in CLDN18.2-positive gastric cancer cells
Given the dual role of autophagy in tumor progression, we aimed to investigate whether autophagy inhibition could potentiate the efficacy of αCLDN18.2-MMAE.In comparison to the administration of αCLDN18.2-MMAEalone, co-administration with the autophagy inhibitor LY294002 resulted in a significantly decreased expression of LC3-II protein and an increased expression of SQSTM1, indicating effective autophagy inhibition.(Fig. 4A, C).As shown in Fig. 4B and D, the combination of αCLDN18.2-MMAE with the autophagy inhibitor LY294002 significantly enhanced its cytotoxicity against MKN45-CLDN18.2and SNU-601 cells.Similarly, coadministration of other autophagy inhibitors such as chloroquine, 3-MA, and hydroxychloroquine also significantly increased the cytotoxic effects of αCLDN18.2-MMAE on these cells (Fig. S3).Furthermore, flow cytometry results showed a substantial augmentation of apoptosis induced by αCLDN18.2-MMAE in MKN45-CLDN18.2and SNU-601 cells in the combination group (Fig. 4E, F).Similarly, combinational treatment increased the cleavage of PARP and caspase-9 (Fig. 4G, H).Therefore, suppressing αCLDN18.2-MMAE-inducedautophagy led to a substantial increase of apoptosis in MKN45-CLDN18.2and SNU-601 cells.These findings uncovered the cytoprotective role of autophagy in αCLDN18.2-MMAE-treatedgastric cancer cells.Combination   5D).Additionally, the combination group displayed heightened expression of cleaved caspase-3 and expression of Ki-67 expression in tumor tissues, indicating an increase in apoptosis and a decrease in proliferation (Fig. 5D).Moreover, H&E staining of heart, liver, spleen, lung, kidney and brain tissues from mice in the experimental group showed no obvious morphological damage caused by drugs compared to the control group (Fig. S4).In conclusion, the in vivo antitumor activity of αCLDN18.2-MMAE was further enhanced by inhibiting autophagy with LY294002, evidenced by augmented apoptosis.
The Akt/mTOR signaling pathway was implicated in the induction of autophagy by αCLDN18.2-MMAETo explore the mechanisms underlying autophagy, we analyzed the alterations in autophagy-related signaling pathways, focusing on changes in the expression levels of key components to elucidate the intrinsic mechanisms of αCLDN18.2-MMAE-inducedautophagy.The Western Blot results demonstrated a dosedependent reduction in phosphorylated mTOR in response to αCLDN18.2-MMAE.Simultaneously, the expression levels of phosphorylated Akt, an upstream signaling protein of mTOR, exhibited a dose-dependent decrease.Furthermore, downstream effectors of Akt/mTOR, including 4E-BP1 and p70S6K, were markedly inhibited (Fig. 6A-D).Given that inhibition of this pathway has been reported to be associated with autophagy activation, these results provided insights into the mechanism underlying autophagy activation induced by αCLDN18.2-MMAE(Fig. 6E).

DISCUSSION
The primary treatment for gastric cancer is surgery and systemic chemotherapy.Unfortunately, the overall prognosis for patients remains poor.To address this challenge, targeted therapies against HER2, CLDN18.2,EGFR, etc., have been developed, with many showing satisfactory results.For example, HER2 is overexpressed in about 20% of gastric cancers.HER2-targeting trastuzumab deruxtecan was approved to treat patients with HER2-positive unresectable or metastatic gastric cancer [2].CLDN18.2 is a highly selective biomarker with low expression in normal tissues and high expression in a broader population suffering from digestive malignancies.In a phase III trial of 2104 patients with locally advanced or metastatic gastric adenocarcinoma, 38.4% of the tumors were determined to be CLDN18.2positive [25].Furthermore, the successful phase III clinical trial of zolbetuximab has proven the efficacy and safety of CLDN18.2 as a potential therapeutic target for gastric cancer.Additionally, CLDN18.2 has demonstrated promising outcomes in ADC therapies, particularly the rapid advancements of IBI343.IBI343 was designed for the treatment of HER2-negative but CLDN18.2-positivegastric cancer and has progressed to a pioneering phase III clinical trial.Despite research into the efficacy of several ADCs targeting CLDN18.2,little headway has been made in elucidating their mechanisms in gastric cancer, underscoring the need and importance of the present study.
Autophagy is a metabolic process that maintains the stability of the intracellular environment and adapts to changes in the external environment.Despite extensive research, the anti-tumor or pro-tumor role of autophagy in gastric cancer remains controversial [26,27].For example, a study reported that the combination of adriamycin and Tanshinone IIA induced autophagy, promoted cell apoptosis, and boosted gastric cancer cell sensitivity to adriamycin [28].On the other hand, activation of autophagy can also yield opposite results.Chemotherapy drugs like 5-fluorouracil and cisplatin were reported to induce cytoprotective autophagy in gastric cancer [29][30][31].As far as we know, there has been no study investigating whether ADCs targeting CLDN18.2induce autophagy and the potential role of autophagy in ADC therapy for gastric cancer.Our study for the first time reveals that CLDN18.2-targetedADC can trigger cytoprotective autophagy in gastric cancer, filling this knowledge gap.While exploring the effect of αCLDN18.2-MMAE on gastric cancer treatment, we observed that MKN45-CLDN18.2and SNU-601 cells formed autophagosomes after treatment with αCLDN18.2-MMAE.Subsequent validation via immunoblotting to evaluate alternations in autophagy-related protein expression, and immunofluorescence to monitor autophagic flux confirmed the induction of autophagy by αCLDN18.2-MMAE.Moreover, our findings indicated that αCLDN18.2-MMAE-inducedautophagy served a cytoprotective role, co-administration of autophagy inhibitor enhanced the antitumor effect of αCLDN18.2-MMAE, as evidenced by increased induction of tumor cell apoptosis and enhanced suppression of tumor growth.
To probe into the mechanism behind αCLDN18.2-MMAEinducedautophagy, we examined various autophagy-related signaling pathways.Specifically, treatment of CLDN18.2-positivegastric cancer cells with αCLDN18.2-MMAEled to a decreased phosphorylation of Akt, mTOR, and two critical downstream effectors, 4E-BP1 and p70S6K [32], indicating the inactivation of the Akt/mTOR pathway.The Akt/mTOR signaling cascade is a classic driver pathway in human cancers [33,34].Once Akt is phosphorylated and activated, it can directly target tuberous sclerosis complex 2 (TSC2), a component of the TSC1/ 2, leading to its inactivation.Consequently, TSC2 loses its inhibitory effect on the Ras homolog enriched in brain (Rheb) protein.Activated Rheb protein binds to mTOR complex 1 (mTORC1), promoting the activation of the mTORC1 signaling pathway [35].Recent research findings have revealed that mTORC1 inhibition not only initiates autophagy but also directly regulates various autophagic processes, including nucleation, elongation, maturation, and termination of autophagosomes [36].Our research further confirmed the involvement of the Akt/mTOR pathway in autophagy induction by αCLDN18.2-MMAE.
In conclusion, our study demonstrated the antitumor efficacy of αCLDN18.2-MMAEagainst CLDN18.2-positivegastric cancer.Importantly, we reported for the first time that CLDN18.2-directedADCs induced cytoprotective autophagy in gastric cancer treatment, with involvement of the Akt/mTOR signaling pathway.Our findings provided theoretical support and experimental evidence for the potential of CLDN18.2-directedADCs, in combination with autophagy inhibitors, to enhance therapeutic efficacy in CLDN18.2-positivegastric cancer.

Cell viability assay
Cells were seeded at a density of approximately 5000 cells per well in 96well plates.After 24 h, the cells were co-incubated with the tested compound for 48 h.The original culture medium was then removed, and each well was replenished with a medium containing 10% Cell Counting Kit-8 (CCK-8) (Beyotime, C0038, Shanghai, China).The cells were then incubated at 37 °C for 1.5 h.After incubation, viable cell absorbance was assessed at 450 nm with a microplate reader.

Transmission electron microscopy analysis
After being treated with or without αCLDN18.2-MMAE(4 μg/mL) for 48 h, MKN45-CLDN18.2and SNU-601 cells were gently collected and subsequently fixed in electron microscopy buffer.The fixed cells were then postfixed in 1% osmium tetroxide for 4 h, rinsed 3 times by phosphate buffer, and dehydrated under gradient ethanol series.Next, the samples were embedded in epoxy resin.Then these resin blocks were sliced, stained, and examined using a transmission electron microscope (Hitachi, HT7800).

Flow cytometry
To assess CLDN18.2expression on the cell surface, cells were harvested and categorized into anti-CLDN18.2antibody and isotype groups.The anti-CLDN18.2antibody group was treated with the primary antibody (DIMA Biotech, DME100179, Wuhan China) and incubated at 4 °C for 30 minutes.After dual PBS washes, a secondary antibody (ABclonal, AS056, Wuhan China) was added, followed by a 30-minute incubation at 4 °C in the dark.After two washes with PBS, cells were resuspended for the next analysis.For analyzing cell apoptosis, MKN45-CLDN18.2and SNU-601 cells were treated with gradient concentrations of αCLDN18.2-MMAE, and Annexin V + /PI + or Annexin V + /PI − cells were detected and classified as apoptotic cells.

Tumor xenograft model
All animal experiments strictly adhered to the guidelines and protocols approved by the Animal Ethical Committee of School of Pharmacy Fudan University.MKN45-CLDN18.2cells were harvested, suspended in PBS, and subcutaneously implanted at a density of 5 × 10^6 cells per NCG mouse.Randomization occurred when the tumor volume reached around 100 mm^3.To assess the antitumor efficacy of αCLDN18.2-MMAE,mice were allocated into three groups receiving PBS, αCLDN18.2-MMAE(2 mg/ kg), or oxaliplatin (5 mg/kg) twice weekly.Regular measurements of tumor dimensions (length and width) were taken.To investigate whether coadministration of the autophagy inhibitor LY294002 enhances the antitumor effects of αCLDN18.2-MMAE,mice were randomly assigned to four groups and received intraperitoneal injections of PBS, αCLDN18.2-MMAE(2 mg/kg), LY294002 (50 mg/kg), or a combination of αCLDN18.2-MMAE and LY294002, twice weekly for 3 weeks.Periodic measurements of tumor dimensions were conducted, and tumor volume was calculated using the formula 1/2 × (long diameter in mm) × (short diameter in mm) × (short diameter in mm).Tumor growth inhibition rate (%) = (1 -(mean volume of treated tumors) / (mean volume of control tumors)) × 100%.

Hematoxylin and Eosin (H&E) staining
Following the acquisition of cellular sections, paraffin removal from the sections was carried out using xylene, followed by a series of alcohol solutions in descending concentrations and ultimately immersion in distilled water.Subsequently, the sections underwent staining with hematoxylin and eosin.Dehydration of the stained sections was achieved with absolute ethanol, followed by rendering the sections transparent with xylene.The processed sections were then mounted, and observations were made under a microscope.

Statistical analysis
The significance of the difference was figured out by Student's t-test or oneway ANOVA with GraphPad Prism 9.0.0.When the P value is more than or equal to 0.05, differences were considered to be not significant and marked as "ns".When the P value is less than 0.05, differences were considered to be statistically different and marked as "*".When the P value is less than 0.01, differences were marked as "**".The results of several independent experiments were displayed as mean ± standard deviations (S.D.).

Fig. 1
Fig. 1 αCLDN18.2-MMAEdemonstrated significant antitumor efficacy against CLDN18.2-positivegastric cancer cells.A Structure of αCLDN18.2-MMAEB The expression level of CLDN18.2 on three human gastric cancer cell lines was determined by flow cytometry.C To evaluate the cytotoxicity of αCLDN18.2-MMAE on CLDN18.2-positivegastric cancer cells, the cell viability was assessed using the CCK-8 assay after treatment with cumulative concentrations of αCLDN18.2-MMAE for 48 h.The results were presented as mean ± S.D. (n = 3) and analyzed by two-tailed unpaired t-test (ns not significant, *P < 0.05, **P < 0.01).D, E Human gastric cancer xenograft models were established by subcutaneous injection of MKN45-CLDN18.2cells into NCG mice.Mice were administered 2 mg/kg of αCLDN18.2-MMAE or 5 mg/kg of oxaliplatin twice weekly.The tumor volume was measured and calculated using length × width 2 /2.The tumor volume data were shown as mean ± S.D. (n = 6).F H&E staining of tumor tissues from the indicated treatment.Scale bar = 20 μm.

Fig. 2
Fig. 2 Apoptosis was activated by αCLDN18.2-MMAE in CLDN18.2-positivegastric cancer cells.A, B MKN45-CLDN18.2 and SNU-601 cells were incubated with indicated concentrations of αCLDN18.2-MMAE for 48 h and then stained with Annexin V-FITC/PI to detect apoptosis by flow cytometry.The proportions of Annexin V-positive cells from repeat experiments were statistically calculated.Data were shown as mean ± S.D. (n = 3) and analyzed by ordinary one-way ANOVA (**P < 0.01).C, D After 48 h of treatment with varying concentrations of αCLDN18.2-MMAE, the expression levels of cleaved PARP and cleaved caspase-9 were determined through Western Blot analysis and quantified by ImageJ.The results were presented as mean ± S.D. (n = 3) and analyzed by ordinary one-way ANOVA (**P < 0.01).